US4990448A - Bu-4061T - Google Patents

Bu-4061T Download PDF

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US4990448A
US4990448A US07/389,479 US38947989A US4990448A US 4990448 A US4990448 A US 4990448A US 38947989 A US38947989 A US 38947989A US 4990448 A US4990448 A US 4990448A
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4061t
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Masataka Konishi
Minoru Hanada
Yuji Nishiyama
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Bristol Myers Co
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Bristol Myers Co
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12RPROCESSES USING MICROORGANISMS
    • C12R1/00Processes using microorganisms
    • C12R1/01Processes using microorganisms using bacteria or actinomycetales
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00Antineoplastic agents
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K5/00Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08Tripeptides
    • C07K5/0802Tripeptides with the first amino acid being neutral
    • C07K5/0804Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0808Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES
    • A61K38/00Medicinal preparations containing peptides
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10STECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00Chemistry: molecular biology and microbiology
    • Y10S435/8215Microorganisms
    • Y10S435/822Microorganisms using bacteria or actinomycetales
    • Y10S435/825Actinomadura

Abstract

A new antibiotic designated BU-4061T is produced by fermentation of actinomycete strain Q996-17 (ATCC-53904). The BU-4061T antibiotic exhibits both in vitro and in vivo antitumor activity.

Description

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention is directed to a new antitumor antibiotic designated herein as BU-4061T and to a process for the preparation, isolation and purification of BU-4061T in substantially pure form.

2. Description of the Prior Art

U.S. patent application Ser. No. 165,337 filed Mar. 7, 1988 discloses a fermentation antitumor antibiotic designated BU-3862T having the structure ##STR1## BU-4061T is somewhat related in structure to the above antibiotic.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 represents the infrared absorption spectrum of BU-4061T (KBr pellet).

FIG. 2 represents the 1 H-NMR spectrum of BU-4061T.

FIG. 3 represents the 13 C-NMR spectrum of BU-4061T.

SUMMARY OF THE INVENTION

This invention relates to a novel antitumor antibiotic designated BU-4061T and to a fermentation process for preparation of BU-4061T using a new actinomycete designated herein as strain Q996-17 (ATCC-53904). The invention also relates to the new microorganism used in the fermentative production of BU-4061T, use of BU-4061T as an antitumor agent and pharmaceutical compositions of BU-4061T adapted for antitumor use.

DETAILED DESCRIPTION OF THE INVENTION THE MICROORGANISM

BU-4061T may be produced by fermentation of actinomycete strain Q996-17 or a BU-4061T-producing variant or mutant thereof.

The preferred producing strain designated Q996-17 was isolated from a soil sample collected in Andhra Pradesh State, India.

The cultural and physiological characteristics of the strain were examined by the methods of Shirling & Gottlieb (Int. J. Syst. Bacteriol. 16:313-340, 1966) and Gordon et al. (J. Gen. Microbiol. 109:69-78, 1978). Diagnostic amino acid and sugar in the whole cell hydrolysate were analyzed by the methods of Lechevalier et al (Biochem. Syst. Ecol. 5:249-260, 1977). The menaquinone samples were prepared by the procedures of Collins et al. (J. Gen. Microbiol. 100:221-230, 1977) and analyzed with a mass spectrometer. The detection of mycolate and the glycolate test were carried out by the methods of Minnikin et al. (J. Gen. Microbiol. 88:200-204, 1975) and Uchida and Aida (J. Gen. Appl. Microbiol. 25:169-183, 1979), respectively.

Morphology: Substrate mycelium is long, well-branched and not fragmented into rods or cocci. Aerial mycelium is not formed in ordinary diagnostic media. Rudimentary aerial mycelium is scarcely born in a part of special media such as rabbit dung agar or malt extract-yeast extract agar supplemented with vitamin B complex. These aerial hyphae turn into a coremium (2-30μm width at base) with tapered tip. Sporulation is not observed on the hyphae tip of coremium or other sites of mycelium.

Cultural and physiological characteristics (Tables 1 and 2): The color of substrate mycelium is grayish-olive in organic media, and colorless or light yellow in chemically defined media. Melanoid pigments are not produced. The growth temperature ranges from 19° C. to 45° C. No growth is observed at 48° C.

Cell chemistry: Whole cell hydrolysate contains meso-diaminopimelic acid, galactose, mannose, ribose and rhamnose and hence the strain belongs to cell wall Type III and sugar pattern C. The phospholipids contain phosphatidylethanolamines, phosphatidylglycerol and phosphatidylinositol; therefore, it is placed in Type P-II. The major menaquinone is MK-9 (H4) and MK-10 (H4). Mycolate is absent. Glycolate test is negative.

Taxonomic position: Strain Q996-17 is an aerobic, mesophilic actinomycete, and does not sporulate. Chemotaxonomically, the strain is related to Streptoalloteichus. Saccharothrix and Actinosynnema. However, the strain cannot be placed in any of the above three genera since it is not characterized with sporulative morphology. Thus, strain Q996-17 is best described as an unidentified actinomycete.

A biologically pure culture of actinomycete strain Q996-17 has been deposited with the American Type Culture Collection, Rockville, Md., U.S.A. and added to its permanent collection of microorganisms as ATCC-53904.

It is to be understood that the present invention is not limited to use of the particular preferred strain or to organisms fully answering its description. It is especially intended to include other BU-4061T-producing variants or mutants of the described organism which can be produced by conventional means such as x-radiation, ultraviolet radiation, treatment with nitrogen mustards, phage exposure, and the like.

ANIBIOTIC PRODUCTION

BU-4061T may be produced by cultivating actinomycete strain Q996-17 (ATCC-53904) or a BU-4061T-producing variant or mutant thereof under submerged aerobic conditions in an aqueous nutrient medium. The organism is grown in a nutrient medium containing an assimilable carbon source, for example, trehalose, D-xylose, D-sorbitol, soluble starch, D-ribose, D-melibiose, D-mannose, D-mannitol, maltose, lactose, D-fructose, glycerol, etc. The nutrient medium should also contain an assimilable nitrogen source such as fish meal, peptone, soybean flour, peanut meal, cottonseed meal, corn steep liquor, yeast extract or ammonium salts. Inorganic salts such as sodium chloride, potassium chloride, magnesium sulfate, calcium carbonate, phosphates, etc. are added if necessary. Trace elements such as copper, manganese, iron, zinc, etc. are added to the medium if desired, or they may be supplied as impurities of other constituents of the media.

Production of BU-4061T can be effected at any temperature conducive to satisfactory growth of the producing organism, e.g. 19°-45° C., but it is preferred to conduct the fermentation at 25°-35° C., most preferably 27°-32° C. Production of the antibiotic is carried out generally for a period of about 3 to 7 days.

The fermentation may be carried out in flasks or in laboratory or industrial fermentors of various capacities. When tank fermentation is to be used, it is desirable to produce a vegetative inoculum in a nutrient broth by inoculating a small volume of the culture medium with a slant or soil culture or a lyophilized culture of the organism. After obtaining an active inoculum in this manner, it is transferred aseptically to the fermentation tank medium for large scale production of BU-4061T. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized in the tank as long as it is such that a good growth of the producing organism is obtained. Agitation during the fermentation can be provided by a mechanical impeller and conventional antifoam agents such as lard oil or silicon oil can be added if needed.

Production of BU-4061T in the fermentation medium can be readily followed during the course of the fermentation by thin layer chromatography or by a cytotoxicity assay.

Isolation of the BU-4061T antibiotic from the fermentation medium and purification of the BU-4061T may be achieved by conventional solvent extraction and chromatographic techniques. A preferred isolation and purification procedure is illustrated in Example 2 below.

PHYSICO-CHEMICAL PROPERTIES OF BU-4061T

BU-4061T was obtained as a white amorphous powder. It was readily soluble in methanol, methylene chloride and ethyl acetate, and practically insoluble in water. BU-4061T gave positive response to iodine vapor, ammonium molybdate-sulfuric acid solution and Rydon-Smith reagent, but negative response to ninhydrin and anthrone reagent. The physico-chemical properties of BU-4061T are summarized in Table 1. The antibiotic did not exhibit characteristic UV absorption. Its IR spectrum in KBr pellet (FIG. 1) showed strong absorptions at 1640 and 1540 cm-1, suggesting that BU-4061T belonged to the peptide group of antibiotics. The 1 H-NMR and 13 C-NMR spectra of BU-4061T are illustrated in FIGS. 2 and 3, respectively. The 13 C-NMR spectrum of BU-4061T demonstrated the presence of 28 carbons including 10 methyl (δ: 10.5, 11.1, 15.5, 15.6, 16.8, 17.8, 21.1, 22.1, 23.3, 32.1), four methylene (24.6, 24.7, 39.5, 52.4), eight methine (25.1, 31.9, 36.2, 50.6, 56.4, 58.0, 61.5, 66.5), one quaternary (59.2) and five carbonyl carbons (170.6, 170.8, 171.7, 172.1, 208.3). The molecular formula of BU-4061T was established as C28 H50 N4 O7 by the 1 H- and 13 C-NMR, microanalysis and SIMS (m/z 577 (M+Na)+, 555 (M+H)+).

STRUCTURAL STUDIES

BU-4061T was hydrolyzed with 6N HCl at 105° C. for 17 hours in a sealed tube. The hydrolyzate was diluted with water and extracted with ether. The separated aqueous layer was concentrated in vacuo and lyophilized to yield 158 mg of colorless oily residue which contained three ninhydrin-positive substances by TLC. Two of them were identical with threonine and isoleucine by their TLC and amino acid analysis behavior. This residue was applied on a column of Dowex 50W×4 (H+ type, 100-200 mesh, φ1.5×20 cm), and the column eluted with 0.03N, 0.06N, 0.1N, 0.3N, 0.6N, 1N and 3N of hydrochloric acid, successively. The pooled ninhydrin-positive fractions eluted with 0:06N HCl were concentrated and further chromatographed on a column of Sephadex LH-20 (φ2.2×100 cm) to yield 12 mg of colorless syrup of threonine hydrochloride. The eluate with 0.3N HCl was concentrated in vacuo to give a mixture of isoleucine and an unidentified ninhydrin-positive substance. Separation of them was achieved by a column chromatography on Dowex 50W×4 (pyridine type, 100-200 mesh, φ2.0×75 cm). The unknown amino acid was eluted with 0.1N pyridine-formic acid buffer (pH 3.1). The eluate was concentrated in vacuo and desalted by Sephadex LH-20 chromatography. Evaporation of the appropriate fractions afforded 12.5 mg of white powder. This substance was determined to be N-methylisoleucine by its SIMS spectrum (m/z:168 (M+Na)+, (146 (M+H)30 ) and 1 H- and 13 C-NMR spectra as shown in Table 2. Isoleucine-containing fractions eluted with 0.2N pyridine-formic acid buffer (pH 3.1) were evaporated in vacuo and the residue was crystallized from aqueous ethanol to give 5.0 mg of colorless needles. The chirality of threonine and isoleucine was determined by using chiral HPLC (TSK gel ENANTIO L1, mobile phase:1 mM CuSO4, detection:254 nm, temperature: 50° C.). The results clearly indicated that both of them has L configuration. The analysis of 1 H- and 13 C-NMR and 1 H--1 H COSY spectrum of BU-4061T (Table 3) exhibited the presence of the following partial structures in addition to L-threonine, L-isoleucine and N-methylisoleucine. ##STR2## The sequence of these fragments was elucidated by analyzing the 13 C-1 H long range COSY and EI-MS spectra of BU-4061T and the structure shown below was assigned to this antibiotic. ##STR3##

              TABLE 1______________________________________Physico-chemical properties of BU-4061T______________________________________Nature         White powderM.P.           107- 109° C.[α].sub.D .sup.24.5          -66.1 ± 0.4 (c 0.5, MEOH)UV λ.sub.max .sup.MeOH nm          end absorptionIR ν.sub.max .sup.KBr cm.sup. -1          3300, 2950, 1720, 1640, 1540SIMS observed m/z          577 (M + Na).sup.+, 555 (M + H).sup.+MicroanalysisCalc'd for C.sub.28 H.sub.50 N.sub.4 O.sub.7          C 60.62      H 9.09  N 10.10Found          C 60.45      H 9.15  N 10.18TLC, SiO.sub.2 CH.sub.2 Cl.sub.2 -MeOH                       9:1     Rf 0.60          Hexane-Acetone                       1:1     0.27______________________________________

              TABLE 2______________________________________.sup.1 H-NMR and .sup.13 C-NMR spectra of N-methylisoleucine ##STR4##           Proton (in D.sub.2 O)           δ(ppm), integration                          Carbon (in D.sub.2 O)Assignment      (multiplicity, J:Hz)                          δ(ppm), multiplicity_________